Analysis of TGF-β/BMP signaling during bone metastasis and development of cancer therapy

转化生长因子-b(转化生长因子-b)超家族成员，包括转化生长因子-β和骨形态发生蛋白，是调节多种细胞反应的多功能细胞因子，包括细胞的增殖、分化、黏附、迁移和凋亡。转化生长因子-β信号通路促进晚期癌症的生长和转移。转化生长因子-β信号通路相应地成为肿瘤学领域药物开发的一个有吸引力的靶点。我们首先建立了一种高度骨转移的人乳腺癌MDA-MB-231细胞变种，命名为MDA-MB-231-5A-D(MDA-231-D)。接下来，我们研究了一种新型的转化生长因子-MDARI型受体(Tbr-I)激酶抑制剂Ki26894对β-231D细胞骨转移的影响。Ki26894通过抑制Smad2的磷酸化和抑制转化生长因子-β反应报告活性的检测，阻断了MDA231-D细胞中的转化生长因子-β信号转导。此外，Ki…26894以上可降低转化生长因子-β诱导的MDA231-D细胞的运动能力和侵袭能力。Ki26894还抑制转化生长因子-β刺激的MDA231-D细胞纤溶酶原激活物抑制物-1、甲状旁腺激素相关蛋白和白介素11mRNA的转录。X线片显示BALB/cnu/nu雌性小鼠左室接种MDA-231-D细胞前1d开始全身应用Ki26894可减少乳腺癌细胞的骨转移。此外，与赋形剂处理的小鼠相比，Ki26894延长了接种了MDA-231-D细胞的小鼠的存活时间。这些发现表明，TβR-I激酶抑制剂如Ki26894可能有助于阻止晚期癌症的进展。此外，我们发现在软骨细胞1(DEC1，也称为SHARP2和Stral3)中差异表达是转化生长因子-β信号转导的下游靶点，从而促进乳腺癌细胞的生存。在小鼠乳腺癌细胞株JygMC(A)和4T1中，Tbr-β激酶抑制剂A-44-03和SB431542在无血清条件下诱导细胞凋亡。寡核苷酸芯片和实时荧光定量逆转录聚合酶链式反应分析显示，转化生长因子-β可诱导DEC1的表达，这种增加可被T-βR-I激酶抑制剂和显性阴性的转化生长因子-βII型受体的表达抑制。DEC1过表达可抑制A-44-03诱导的JygMC(A)细胞的凋亡，下调内源性DEC1可抑制转化生长因子-β促进细胞存活。此外，DEC1的显性-负性突变体在体内阻止了JygMC(A)细胞的肺和肝转移。因此，我们的观察为研究转化生长因子-β介导的癌细胞存活和转移的分子机制提供了新的见解。较少

Members of the transforming growth factor-b (TGF-β) superfamily, including TGF-β and bone morphogenetic proteins (BMPs), are multifunctional cytokines that regulate a wide range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis. TGF-β signaling facilitates tumor growth and metastasis in advanced cancer. The TGF-β signaling pathway has correspondingly become an attractive target for drug development in the field of oncology.We investigated the roles of TGF-β and BMP signaling during bone metastasis. We first established a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Next, we examined the effects of a novel TGF-β type I receptor (TBR-I) kinase inhibitor, Ki26894, on bone metastasis of MDA-231-D cells. Ki26894 blocked TGF-β signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-β-responsive reporter activity. Moreover, Ki … More 26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-β in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-β. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/c nu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that TβR-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.In addition, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stral3) as a downstream target of TGF-β signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC (A) and 4T1, the TBR-β kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-β induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TβR-I kinase inhibitors as well as by expression of dominant-negative TGF-β type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC (A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-β-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC (A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-β-mediated cell survival and metastasis of cancer. Less